Vitamin D and its analogs in the treatment of tumors and other hyperproliferative disorders

ABSTRACT

Treatment of hyperproliferative disorders (including tumors and psoriasis) by pulse administration of a drug (such as Vitamin D or an analog) that increases blood or tissue levels of Vitamin D. The drug is administered at a sufficient dose to have an anti-proliferative effect, but the pulsed administration of the drug avoids the development of severe symptomatic or life-threatening hypercalcemia. In particular embodiments, avoidance of hypercalcemia (as measured by serum levels of calcium above normal range) is avoided altogether. In a particular example, the drug is calcitriol administered at an oral dose of about 0.5 mcg/kg once a week.

FIELD OF THE INVENTION

[0001] This invention concerns the use of Vitamin D and its analogs inthe treatment of tumors and hyperproliferative disorders.

BACKGROUND OF THE INVENTION

[0002] Vitamin D is a generic term for a family of secosteroids thathave affinity for the Vitamin D receptor, and are involved in thephysiologic regulation of calcium and phosphate metabolism. Exposure tothe sun and dietary intake are common sources of Vitamin D, butdeficiencies of this vitamin can cause rickets and osteomalacia.Supplementation of dairy and other food products has reduced theincidence of Vitamin D deficiency conditions in modem society, andmedical research concerning this vitamin has turned to its therapeuticeffects in a variety of pathological conditions.

[0003] Vitamin D₃ is synthesized in human skin from 7-dehydrocholesteroland ultraviolet light. Vitamin D₃, or its analog Vitamin D₂, can beingested from the diet, for example in fortified milk products. VitaminD₂ and D₃ undergo hydroxylation first in the liver to 25-hydroxyvitaminD, then in the kidney to 1α,25-dihydroxycholecalciferol (also known as1,25-dihydroxyvitamin D or calcitriol), which is the principalbiologically active form of Vitamin D. The biological production of thisactive form of the vitamin is tightly physiologically regulated.

[0004] Vitamin D exerts its calcium regulating activity through bothgenomic and nongenomic pathways. Although the nongenomic pathways remainpoorly characterized, the genomic responses are mediated through bindingto the nuclear Vitamin D receptor (VDR). The VDR is a ligand-activatedtranscription factor, which binds the Vitamin D₃ response elementcontained within the promoter/enhancer region of target genes. Vitamin Dmaintains calcium levels in the normal range by stimulating intestinalcalcium absorption. When intestinal absorption is unable to maintaincalcium homeostasis, Vitamin D stimulates monocytic cells to becomemature osteoclasts, which in turn mobilize calcium from bones.

[0005] Appreciation for Vitamin D's non calcium-related biologicalactivities began in 1979 with Stumpf's discovery that radioactiveVitamin D localizes to many tissues not associated with calciummetabolism (Science 206:1188-1190, 1979). In 1981, Abe et al. reportedthat mouse myeloid leukemia cells possessed VDR, and that their exposureto Vitamin D led to terminal differentiation (PNAS USA 78:4990-4994,1981). Since then VDR has been described in carcinomas of the prostate,breast, colon, lung, pancreas, endometrium, bladder, cervix, ovaries,squamous cell carcinoma, renal cell carcinoma, myeloid and lymphocyticleukemia, medullary thyroid carcinoma, melanoma, multiple myeloma,retinoblastoma, and sarcomas of the soft tissues and bone.

[0006] In vitro assays using 1,25 dihydroxyvitamin D or its analoguesdemonstrated antiproliferative effects in cell lines derived from manymalignancies including adenocarcinomas of the prostate (Molec. and Cell.Endocrinology 126:83-90, 1997; Proc. Amer. Assoc. Cancer Res. 38:456,1997; J. Ster. Biochem. and Molec. Biol. 58:277-288, 1996; Endocrinology137:1554-1561, 1996; Endocrinology 136:20-26, 1995; Cancer Research54:805-810, 1994; Endocrinology 132:1952-1960, 1993; and AnticancerResearch 14:1077-1081, 1994), breast (Proc. Amer. Assoc. Cancer Res.38:456, 1997; Biochemical Pharmacology 44:693-702, 1992); colon(Biochemical and Biophysical Research Communications 179:57-62, 1991;Archives of Pharmacology 347:105-110, 1993); pancreas (British Journalof Cancer 73:1341-1346, 1996); and endometrium (Journal of Obstetricsand Gynaecology Research 22:529-539, 1996); lung (Anticancer Research16:2953-2659, 1996); myeloid leukemia (PNAS USA 78:4990-4994, 1981);melanoma (Endocrinology 108:1083-1086, 1981); and sarcomas of the softtissues (Annals of Surgical Oncology 3:144-149, 1996) and bone (Journalof the Japanese Orthopaedic Association 69:181-190, 1995).

[0007] Studies in animals have shown antiproliferative activity ofVitamin D or its analogues in prostate cancer (Urology 46:365-369,1994); breast cancer (J. NCI 89:212-218, 1997; Lancet 1: 188-191, 1989);squamous cell carcinoma (Molecular and Cellular Differentiation 3:31-50,1995); myeloid leukemia (Blood 74:82-93, 1989 and PNAS USA 80:201-204,1983) and retinoblastoma (Archives of Opthalmology 106:541-543, 1988;Archives of Opthalmology 106:536-540, 1988). The mechanism of VitaminD's antiproliferative effects remains unknown, although it has beenproposed that Vitamin D increases synthesis of TGF-β1 and TGF-β2,decreases the expression of epidermal growth factor receptors, leads todephosphorylation of the retinoblastoma protein, induces cell cyclearrest in G1, perhaps by induction of the cyclin dependent kinaseinhibitors p21 (waf1) and p27 (kip1), and induces the production ofinsulin-like growth factor binding protein.

[0008] The patent literature is replete with attempts to treat tumorswith Vitamin D compounds. U.S. Pat. No. 4,391,802 disclosed treatingleukemioid diseases with 1α-hydroxy Vitamin D derivatives. The use of1α-hydroxy derivatives with a 17 side chain greater in length than thecholesterol or ergosterol side chains was disclosed in U.S. Pat. No.4,717,721. Additional Vitamin D analogs are described in U.S. Pat. No.4,851,401 (cyclopentano-Vitamin D analogs), U.S. Pat. No. 4,866,048,U.S. Pat. No. 5,145,846 (Vitamin D₃ analogs with alkynyl, alkenyl, andalkanyl side chains), U.S. Pat. No. 5,120,722 (trihydroxycalciferol),U.S. Pat. No. 5,547,947 (fluorocholecalciferol compounds), U.S. Pat. No.5,446,035 (methyl substituted Vitamin D), U.S. Pat. No. 5,411,949(23-oxa-derivatives), U.S. Pat. No. 5,237,110 (19-Nor-Vitamin Dcompounds), U.S. Pat. No. 4,857,518 (hydroxylated 24-homo-Vitamin Dderivatives). Additional Vitamin D analogs are shown in U.S. Pat. Nos.4,804,502; 5,374,629; 5,403,940; 5,446,034; and 5,447,924.

[0009] Few attempts have been made to test Vitamin D's antiproliferativeeffects in humans with cancer. Koeffler et al., Cancer Treatment Reports69:1399-1407, 1985, gave 2 mcg of 1,25-dihydroxyvitamin D daily for 8weeks or longer to 18 patients with myelodysplastic syndrome. Eight of18 patients had minor and transient improvements in the peripheral bloodcounts, but by the end of the 12 week study no patient showedsignificant improvement and 4 patients experienced symptomatichypercalcemia. Bower et al., Lancet 337:701-702, 1991, treated 19patients with locally advanced or cutaneous metastatic breast cancerwith topical calcipotriol, a Vitamin D analogue. Three of the 14patients who completed 6 weeks of treatment showed a 50% reduction inthe bidirectional diameter of the treated lesions and one other patientshowed minimal response, however hypercalcemia was a complication of thetreatment. Palmieri-Sevier et al., Am. J. Medical Sciences 306:309-312,1993, reported a case of long term remission of parathyroid carcinomawhich appeared to be induced and maintained by Vitamin D therapy. Rustinet al., Brit. J. Can. 74:1479-1481, 1996, performed a clinical trialwith a continuous dose of calcitriol in patients with ovarian cancer,and again encountered hypercalcemia.

[0010] A phase II trial of oral 1,25-dihydroxyvitamin D (calcitriol) inhormone refractory prostate cancer was reported by Osborn et al., Urol.Oncol., 1: 195-198, 1995. Fourteen patients were given a daily oral doseof 0.5-1.5 mcg calcitriol, but no significant response was demonstrated,and clinical deterioration was documented in most of the patients.Thirteen of the patients experienced hypercalcemia, which is the mostcommon side effect of treatment with Vitamin D and its analogs. Concernthat hypercalcemic effects of Vitamin D would preclude the achievementof therapeutic, anti-neoplastic serum levels has inhibited the study ofthe use of this vitamin in humans with cancer. It is an object of thisinvention to provide a method of treatment with Vitamin D drugs (such ascalcitriol) that avoids such hypercalcemia, while permitting the use ofthis class of drugs in the treatment of tumors and otherhyperproliferative diseases.

SUMMARY OF THE INVENTION

[0011] Vitamin D and its analogs can be administered in accordance withthe present invention, for the treatment of neoplastic diseases, such asthe types of tumors mentioned above, which are responsive to treatmentwith Vitamin D drugs. The method can also be used to treathyperproliferative skin diseases, such as psoriasis, disorders ofkeratinization and keratosis, or disorders of sebaceous glands, such asacne or seborrheic dermatitis. The method includes administering to thesubject a therapeutically effective pulsed dose of the Vitamin D drug ina sufficient amount to have a therapeutic effect, without inducinghypercalcemia, particularly symptomatic hypercalcemia, for example stage3 or stage 4 hypercalcemia. This treatment is especially effective toallow the use of highly calcemic drugs (such as drugs having a calcemicindex of 0.5 or more) which are often highly effective in the treatmentof tumors and hyperproliferative diseases, but which have been avoidedin the past because of their calcemic side effects. The dosing regimenof the present invention for the first time allows therapeuticallyeffective antiproliferative (and particularly antineoplastic) amounts ofthese drugs to be given without inducing the dangerous side effect oflife-threatening hypercalcemia, while surprisingly having a prolongedtherapeutic specific anti-tumor or general antiproliferative effect.

[0012] In a first disclosed embodiment, the Vitamin D drug isadministered to a subject having a neoplasm that expresses a Vitamin Dreceptor, and responds to treatment with a Vitamin D drug. Particulartypes of tumor that respond to such treatment include adenocarcinomas ofthe prostate, breast, colon, pancreas and endometrium, as well as smallcell and non-small cell cancer of the lung (including squamous,adenocarcinoma and large cell types), squamous cell carcinoma of thehead and neck, transitional cell cancer of the bladder, ovarian andcervical (e.g. squamous cell carcinoma) cancer, renal cell carcinoma,myeloid and lymphocytic leukemia, lymphoma, medullary thyroid carcinoma,melanoma, multiple myeloma, retinoblastoma, and sarcomas of the softtissues and bone. In particular embodiments, the neoplasm isadenocarcinoma of the breast or prostate.

[0013] In yet other specific embodiments, the Vitamin D drug is one thatwould induce hypercalcemia (particularly symptomatic or life-threateninghypercalcemia) in a subject to which the drug is given atantiproliferative doses. The method would have particular application todrugs that are as calcemic as calcipotriol (calcemic index of about0.005-0.01), 11α-fluoromethyl-1α,25-(OH)₂-D₃ (having a calcemic index ofabout 0.1), and drugs having a calcemic index greater than 0.5, forexample greater than or equal to 1 (the calcemic index of calcitriol).Drugs with which the method is particularly useful are those drugshaving a half-life no greater than about 1 day, for example no greaterthan about 6 hours, when the dose is given as a therapeuticallyeffective dose. These half-lives are sufficiently short that they allowthe blood level to return to non-calcemic levels for a sufficient periodbetween doses so that full osteoclast activation does not occur. Inparticular embodiments, blood levels of calcium return to normal betweendoses. The Vitamin D drug is administered in an amount that raises aserum level of Vitamin D in the subject with a tumor to asupraphysiologic amount for a sufficient period of time to inducedifferentiation or regression of the tumor without causing symptomatichypercalcemia.

[0014] For example, where the Vitamin D analog is calcitriol, it can beadministered in a high pulse dose no more than once every three days,for example once a week. Although calcitriol has been used in the pastto treat cancer, dosages of such regimens have been 0.5-1.5 mcg per dayfor prolonged periods of time, which has caused symptomatichypercalcemia. In accordance with some embodiments of the presentinvention, calcitriol is orally administered in a dose of at least 0.12mcg/kg per day (8.4 mcg in a 70 kg person) no more than once every 5 or6 days, for example once a week. Even higher doses of calcitriol arepossible using the pulsed dose regimen of the present invention, forexample administering the calcitriol orally in a dose of about at leastabout 0.48 mcg/kg per day, for example 1 mg/kg per day or higher, e.g.2-3 mg/kg per day, no more than once per week. As the dosage of thecalcitriol or other Vitamin D drug increases, the interval between dosescan be increased (for example to as long as 7-10 days) to avoidsymptomatic hypercalcemia. It has surprisingly been observed that pulsedincreases in the blood level of Vitamin D are sufficient to have ananti-tumor or antiproliferative effect for a prolonged period of time(e.g. 10 days), so that the dosing regimen of the present invention canbe followed while encountering a lowered risk of hypercalcemia.

[0015] The invention also includes a pharmaceutical compositioncomprising a Vitamin D drug in a pharmaceutical dosage form containingat least 5 micrograms (mcg) of calcitriol, for example 5-100 mcg. Thedosage form may be an oral, intravenous, intramuscular, topical,subcutaneous, transdermal, sublingual, intranasal, intratumoral or otherpreparation, but in particular disclosed embodiments the pharmaceuticaldosage form is an oral dosage form, such as a tablet or capsule.

[0016] The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription of several preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 is a diagram showing peak and trough plasma calcitriollevels in subjects who received the indicated dose of calcitriol overfour hours. Peak levels (⋄) were determined at 6 hours afteradministration, and trough levels (◯) were determined at 48 hours afteradministration.

[0018]FIG. 2 shows a time course of plasma calcitriol levels in asubject who received a 2.0 μg/kg dose of calcitriol.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

[0019] Definitions The following definitions will help with anunderstanding of the terms used in this specification.

[0020] A “Vitamin D drug” is a drug that raises the blood or tissuelevel of Vitamin D, or has an affinity for the Vitamin D receptor, forexample binding to that receptor with a Relative Competitive Index (RCI)of 0.05 or greater, more particularly 5 or greater, for example 5-250.The RCI is indexed to an RCI of 100 for calcitriol. The term alsoincludes any of the family of secosteroids with antirhichitic activity,such as Vitamin D₂ (ergocalciferol) and Vitamin D₃ (cholecalciferol),their precursor molecules such as ergosterol(7-dehydro-22-dehydro-24-methyl-cholesterol) and 7 dehydrocholesterol,25-hydroxyvitamin D₃ the 3-hydroxylated dihydrotachysterol₂, the1α-hydroxylated alfacalcidol (1α-hydroxyvitamin D₃) and calcitriol(1α,25-dihydroxyvitamin D₃), as well as the numerous natural andsynthetic Vitamin D analogs set forth in the attached Appendix I (fromBouillon et. al, Endocrine Reviews 16: 200-257,1995).

[0021] Vitamin D drugs also include Vitamin D preparations and analogsthat are currently in clinical use, such as Rocaltrol® (RocheLaboratories), Calcijex® injectable calcitriol, investigational drugsfrom Leo Pharmaceutical including EB 1089(24a,26a,27a-trihomo-22,24-diene-1αa,25-(OH)₂-D₃), KH 1060(20-epi-22-oxa-24a,26a,27a-trihomo-1α,25-(OH)₂-D₃), MC 1288 and MC 903(calcipotriol), Roche Pharmaceutical drugs that include1,25-(OH)₂-16-ene-D₃, 1,25-(OH)₂-16-ene-23-yne-D₃, and25-(OH)2-16-ene-23-yne-D₃, Chugai Pharmaceuticals 22-oxacalcitriol(22-oxa-1α,25-(OH)2-D₃; 1α-(OH)D₅ from the University of Illinois; anddrugs from the Institute of Medical Chemistry-Schering AG that includeZK 161422 and ZK 157202. Appendix 3 provides additional informationabout chemical structure, route of administration and dosing of some ofthese compounds. Vitamin D analogs also include topical preparations ofVitamin D, such as Calcipotriene (Dovonex) and Tacalcitol, used in thetreatment of psoriasis.

[0022] A “Vitamin D receptor” (or VDR) is a protein transcriptionfactor, for which the gene and its product have already beencharacterized and found to contain 427 amino acids, with a molecularweight of about 47,000, or variants thereof The full length cDNA of thehuman VDR is disclosed in Baker et al., PNAS, USA 85:3294-3298, 1988.

[0023] “Tumor cells that express (or contain) the Vitamin D receptor”are those tumors that have been shown to contain the Vitamin D receptor,tumors that are subsequently shown to contain the receptor (usingimmunohistochemical or other techniques), tumor types (such as breastcancer) that have demonstrated a clinical improvement in response totreatment with calcitriol or its analogs or other Vitamin D drugs, andtumors for which there is epidemiologic data demonstrating anassociation between low Vitamin D levels and higher cancer incidence(such as adenocarcinomas of the prostate, breast and colorectum). Thepresence of Vitamin D receptors can be determined by any means known inthe art, such as any of the techniques disclosed in Pike, Ann Rev. Nut.11: 189-216, 1991.

[0024] “Elevated (or supraphysiologic) level of Vitamin D” refers to a1,25-dihydroxyvitamin D plasma concentration greater than about 0.15 nm(65 pg/ml), or other Vitamin D concentration greater than normal in thelaboratory where the concentration is measured, for example in humans atotal human plasma concentration greater than about 10 ng/ml of25-hydroxyvitamin D (although this and all other normal values can varydepending on the techniques used to measure the concentration).

[0025] “Hypercalcemia” refers to a calcium plasma concentration greaterthan normal in the laboratory where the concentration is measured, forexample greater than about 10.5 mg/dL in humans (although this and allother normal values can vary depending on the techniques used to measurethe concentration). Hypercalcemia can be broken into grades 0-4, as setforth in Appendix II.

[0026] “Symptomatic hypercalcemia” refers to laboratory demonstratedhypercalcemia associated with one of more of the signs or symptoms ofhypercalcemia. Early manifestations of hypercalcemia include weakness,headache, somnolence, nausea, vomiting, dry mouth, constipation, musclepain, bone pain, or metallic taste. Late manifestations includepolydypsia, polyuria, weight loss, pancreatitis, photophobia, pruritis,renal dysfunction, aminotransferase elevation, hypertension, cardiacarrhythmias, psychosis, stupor, or coma. Ectopic calcification has beenreported when the calcium-phosphate product (multiplying theconcentrations of calcium and phosphate) exceeds 70. “Severe symptomatichypercalcemia” refers to grade 3 or grade 4 hypercalcemia.

[0027] A “tumor” is a neoplasm, and includes both solid and non-solidtumors (such as hematologic malignancies). A “hyperproliferativedisease” is a disorder characterized by abnormal proliferation of cells,and generically includes skin disorders such as psoriasis as well asbenign and malignant tumors of all organ systems. “Differentiation”refers to the process by which cells become more specialized to performbiological functions, and differentiation is a property that is totallyor partially lost by cells that have undergone malignant transformation.

[0028] A “therapeutically effective dose” is a dose which in susceptiblesubjects is sufficient to prevent advancement, or to cause regression ofthe disease, or which is capable of relieving symptoms caused by thedisease, such as fever, pain, decreased appetite or chachexia associatedwith malignancy.

[0029] A “pulse” dose of a Vitamin D drug refers to administration ofthe drug in a sufficient amount to increase the blood or tissue level ofVitamin D to a supraphysiologic concentration for a sufficient period oftime to have a therapeutic benefit, but with a sufficient period betweendoses to avoid hypercalcemia, given the pharmacological half life of thedrug, its rate of elimination from the body, and its calcemic index.

[0030] The “calcemic index” of a drug is a measure of the relativeability of a drug to generate a calcemic response, for example asmeasured and reported in Bouillon et al., Endocrine Reviews 16:200-257,1995. A calcemic index of 1 corresponds to the relative calcemicactivity of calcitriol. A calcemic index of about 0.01 corresponds tothe calcemic activity of calcipotriol. A calcemic index of 0.5 wouldcorrespond to a drug having approximately half the calcemic activity ofcalcitriol. The calcemic index of a drug can vary depending on the assayconducted, e.g. whether measuring stimulation of intestinal calciumabsorption (ICA) or bone calcium mobilizing activity (BCM), as reportedin Hurwitz et al., J. Nutr 91:319-323, 1967 and Yamada et al.,Molecular, Cellular and Clinical Endocrinology (Berlin), pages 767-774,1988. Hence relative calcemic activity is best expressed in relation tothe calcemic activity of calcitriol, which is one of the bestcharacterized Vitamin D drugs.

Vitamin D Drugs

[0031] Normal serum levels of 1,25-dihydroxyvitamin D range between 0.05and 0.16 nM, however therapeutic drug levels necessary for cancerinhibition have not been well defined. Skowronski et al. (Endocrinology136-20-26, 1995) demonstrated measurable growth inhibition of LNCaPhuman prostate cancer cells in vitro at a 1,25-dihydroxyvitamin Dconcentration of 0.1 nM and 50% growth inhibition at a 1.0 nMconcentration. Peehl et al. (Cancer Research 54:805-810, 1994) incubatedhuman prostate cancer cells in primary culture with1,25-dihydroxyvitamin D concentrations ranging between 0.025 and 25 nMand demonstrated half maximal growth inhibition at levels between 0.25and 1.0 nM. Previous clinical trials of Vitamin D in the treatment ofcancer have proceeded on the assumption that high levels of the drugwere needed for a prolonged period of time to have a therapeuticbenefit. The inventors of the present invention, however, havesurprisingly shown that intermittent supraphysiologic levels of1,25-dihydroxyvitamin D (for example greater than or equal to 0.25 nM)are sufficient to inhibit cancer growth and other proliferative diseasein mammals. This surprising finding now permits the therapeutic benefitsof Vitamin D therapy to be achieved without substantial risk ofmorbidity from iatrogenic hypercalcemia induced by the therapy.

[0032] Calcitriol is a short acting preparation of 1,25-dihydroxyvitaminD, which therefore offers an opportunity for intermittent treatmentaimed at achieving high serum 1,25-dihydroxyvitamin D levels for briefperiods of time. This regimen has surprising anti-tumor activity, whileminimizing toxicity, such as hypercalcemia. Calcitriol has primarilybeen studied when chronically administered as replacement therapy, forwhich its usual dose is 0.25-1.0 mcg per day. Peak serum concentrationis reached at 2 hours and serum half life is 3-6 hours. Intestinalabsorption of calcium begins to increase 2 hours after administration.Hypercalcemic effect is maximal at 10 hours and lasts 3-5 days.

[0033] In one embodiment of the invention, a sufficient dose ofcalcitriol is administered to raise serum 1,25-hydroxyvitamin D levelsto a therapeutically effective level for a pulsed dose that has ananti-proliferative effect without causing significant hypercalcemia (forexample symptomatic grade 3 or grade 4 hypercalcemia). With calcitriol,an example of such a dose would produce a serum level of at least about0.5 nM, for example about 0.9 nM or more (e.g. 1-25 nM, for example 5-10nM), for at least 2 hours (e.g. 2-5 hours) and preferably no more than 6hours. In particular embodiments, the pulsed dose of calcitriol does notexceed a dose at which symptomatic hypercalcemia occurs, or morepreferably a pulsed dose at which even laboratory hypercalcemia occurs.

[0034] Information about short term effects of higher than replacementdoses of calcitriol is available for helping predict drug effects.Papapoulus et al., (Clinical Science 62:427-429, 1982) gave 2 mcg ofcalcitriol as a single oral dose to healthy volunteers and achieved peak1,25-dihydroxyvitamin D serum concentrations of 0.235 and 0.351 nM.Mason et al. (BMJ 1980:449-450) gave a single oral dose of 4 mcgcalcitriol to healthy volunteers and achieved peak 1,25-dihydroxyvitaminD serum concentrations of 0.42 nM with no elevation in serum calcium.Brickman et al. (Am J. Med. 57:28-33, 1974) treated normal volunteerswith calcitriol doses up to 2.7 mcg/day for 7 to 15 days. While calciumabsorption and excretion were increased, no significant elevations inserum calcium were observed. Adams et al. (Kidney Int. 21:90-97, 1982)treated normal volunteers with up to 3 mcg/day of calcitriol for 6-12days and achieved stable 1,25-dihydroxyvitamin D serum levels of0.184-0.235 nM. None of the patients who were on a low calcium dietexperienced elevation in serum calcium. Geusens et al. (Calcified TissueInt. 49:168-173, 1991) gave 4 mcg of calcitriol per day for 4 days to 27postmenapausal women with osteoporosis or osteoarthritis. Theydemonstrated increased urinary calcium excretion but no increase inurinary hydroxyproline excretion. Four of the 27 patients had a serumcalcium above 10.8 but no clinically significant hypercalcemia wasobserved.

[0035] Antiproliferative levels of 1,25-dihydroxyvitamin D can beachieved for short periods of time with minimal adverse effects,particularly if hypercalcemia during short course 1,25-dihydroxyvitaminD therapy is primarily mediated by increases in intestinal calciumabsorption (slower calcium elevation) rather then osteoclast activation(which can rapidly mobilize calcium from bone). Higher1,25-dihydroxyvitamin D levels are achievable when the drug is given inconjunction with a reduced calcium diet to minimize intestinal calciumabsorption, and adequate hydration to maximize calcium excretion. Themaximal tolerated dose of calcitriol, when given intermittently has notbeen defined, but doses as high as 0.48 mcg/kg have been toleratedwithout hypercalcemia.

[0036] Higher doses of a Vitamin D drug, sufficient to achievetherapeutic antiproliferative levels, may also be achieved byadministering the drug in conjunction with bisphosphonate osteoclastinhibitors, such as pamidronate. Selby et al. (Endocrinology108:1083-1086, 1981) provided an example of treating hypercalcemia dueto Vitamin D with pamidronate. The potential for achieving high serum1,25-dihydroxyvitamin D levels when osteoclasts are inhibited inpatients with osteopetrosis is possible with calcitriol doses as high as32 mcg/day for 3 months (Key et al., NEJM 310:409-415, 1984) wherestable serum levels of 1,25-dihydroxyvitamin D peaked at 2.32 nM with nohypercalcemia.

[0037] The following Examples illustrate the general method of thepresent invention, as well as specific case histories to illustrate itsuse. These Examples also provide a general framework for evaluatingother Vitamin D drugs, and determining a therapeutically effective doseof a Vitamin D drug in a subject with a Vitamin D responsivehyperproliferative disease, without inducing symptomatic iatrogenichypercalcemia.

EXAMPLE 1 General Treatment Plan

[0038] A patient with a known Vitamin D receptor positive tumor (such asadenocarcinoma of the prostate, breast, lung, colon or pancreas, ortransitional cell carcinoma of the bladder, or melanoma) may be placedon a prescribed reduced calcium diet prior to treatment, to helpminimize intestinal absorption and allow even higher doses of theVitamin D drug to be used. This reduced calcium diet may be continuedfor the duration of treatment, and for one week after the last dose ofthe Vitamin D drug. The diet ideally restricts daily calcium intake to400-500 mg, by avoiding all dairy products, as well as sardines andother fish canned with their bones, legumes, greens, and any calciumfortified foods or drinks. The subject is then asked to drink 4-6 cupsof fluid more than usual intake starting 12 hours before treatment andcontinuing for days 1, 2, and 3, to assure adequate oral hydration.Magnesium containing antacids, oral calcium supplements, cholestyramine,colestipol, and other bile resin binding agents may also be avoidedduring treatment.

[0039] Baseline laboratory tests that may be obtained include serumlevels of calcium, phosphate, and 1,25-dihydroxyvitamin D. At theinitial dose level, e.g. calcitriol 0.06 mcg/kg po (or another Vitamin Ddrug for which the dose is to be determined) is divided into 4 doses,and one of those four doses is taken during each hour for 4 hours untilthe total 0.06 mcg/kg dose is taken. Alternatively, a single higher doseformulation may be ingested. The doses may be rounded up to the nearest0.5 mcg. The subject is monitored daily for symptoms of hypercalcemiafor at least 2-3 days following administration.

[0040] The patient may have a variety of laboratory tests performed tomonitor the presence of hypercalcemia, or any physiological consequencesof hypercalcemia. Such tests may include calcium at 0, 24, 48 hours, andbaseline levels of creatinine, total billirubin, ALT, alkalinephosphatase, and a complete blood count. Other possible laboratory testsinclude phosphate, 1,25-dihydroxyvitamin D levels at 0, 6, 24, 48 hours,and 24 hour urine collection for calcium and hydroxyproline on day 2.Subjects are treated with the once a week pulse dose of Vitamin D weeklyuntil disease progression or 4 weeks, whichever comes first, and arefollowed for 2 months from enrollment. If Grade 3 toxicity isencountered, the treatment is stopped.

[0041] An initial dose may be chosen from safe doses documented in theliterature, followed by a multistage escalation scheme, such as the onedescribed by Gordon and Willson (Statistic in Medicine 11:2063-2075,1992). Patient accrual occurs in stages I, II, and III. The stagesrequire the accrual of one, three, or six patients respectively beforedose escalation. All patients enrolled at a dose level complete 4 weeksof treatment before the dose level is escalated. In stage I, a singlepatient is entered at each dose level. Accrual continues in stage Iuntil the first Grade 3 toxicity is encountered. When a Grade 3 toxicityis encountered, two more patients are accrued at the same dose level andaccrual will continue in stage II. Doses are reduced one level if oneGrade 4 or 5 toxicity is encountered in stage I.

[0042] Accrual continues in stage II if no Grade 3 toxicities areencountered. When one or two Grade 3 or Grade 4 toxicities areencountered, three additional patients are accrued at the same doselevel and accrual continues in stage III. Doses are reduced one level ifone Grade 5 or three Grade 3 or Grade 4 toxicities are encountered instage II. In stage III six patients are enrolled at each dose level. Ifonly one Grade 3 toxicity is encountered, the dose will be escalated andthe accrual will revert to stage II. If two or more Grade 3 or greatertoxicities occur, no further escalation will occur. The MTD (maximumtolerated dose) is defined as that dose at which ⅓ or fewer of thesubjects experience grade 3 toxicity. For calcitriol, the initial dosewas 0.06 mcg/kg po over 4 hours. At each successive level, the dose isdoubled until the first grade 3 toxicity is encountered. After that,each dose increase is 1.33×of the preceding level according to amodified Fibonacci scheme (Diliman and Koziol, Molecular Biotherapy4:117-121, 1992).

[0043] For calcitriol, the pulse dose was given to each subject weekly,and the subject was monitored for early signs and symptoms ofhypercalcemia, such as weakness, headache, somnolence, nausea, vomiting,dry mouth, constipation, muscle pain, bone pain, metallic taste. Thepatient was also monitored for any more serious manifestations, such aspolydypsia, polyuria, weight loss, pancreatitis, photophobia, pruritis,renal dysfunction, aminotransferase elevation, hypertension, cardiacarrhythmias, psychosis, stupor, coma, and ectopic calcification.Appropriate treatment is instituted for any patient who demonstrateshypercalcemic toxicity, and the calcitriol is stopped until serumcalcium returns to normal.

[0044] The following Table 1 illustrates a protocol that can be followedwith each drug to determine a tolerated pulse dose. A protocol fordetermining a therapeutic dose will be described in Example 2. TABLE 1Example of Protocol for Determining Tolerated Dose WEEKS 5-8 WEEKS 1-4Follow-Up Premature EVALUATION Day Day Day Week Week Until Ca nl AfterCa nl Evaluation & Procedures Screen 1 2 3 5 7 daily every 2 InformedConsent x Inclusion/Exclusion Criteria x Physical Exam x x x x SittingVital Signs x x x Adverse Effects Recorded x x x x x x Calcitrioladministered x Calcium x x x x x x x x Phosphate x x x x x1,25-dihydroxyvitamin D level x xx x x x Creatinine x x x TotalBillirubin x x x ALT x x x Akaline Phosphatase x x x Albumin x CompleteBlood Count x x β-hCG (select patients) x Urine Collection x x DietQuestionnaire x Tumor measurements (when x x appropriate)

EXAMPLE 2 Determination of Therapeutically Effective Dose

[0045] Tumor markers, such as PSA, CA 15-3, and others can be used toassess tumor progression or regression, although the results of suchassays can sometimes be difficult to interpret because administration ofVitamin D has been shown to increase tumor marker production whileinhibiting cancer cell growth. This effect is presumably due to thedifferentiation inducing properties of Vitamin D.

[0046] Alternative means for determining a therapeutic response can alsobe employed, for example direct radiographic measurement of tumorlesions. A measurable lesion may be considered one that isbidimensionally measurable, with clearly defined margins on physicalexams, x-ray, or scan. At least one diameter is preferably greater than0.5 cm. Bone lesions are not included.

[0047] Evaluable disease includes unidimensionally measurable lesions,masses with margins not clearly defined, palpable nodal disease, lesionswith both diameters less than 0.5 cm, and bone disease. Non-evaluabledisease includes disease manifested by pleural effusions, ascites, ordisease documented by indirect evidence only (e.g., by lab values whichfall into a category of not being evaluable). The objective status isrecorded at entry into the trial and during week 7 (where week 1 is theweek during which the first dose of the Vitamin D drug is given). If anorgan has too many measurable lesions to measure at each evaluation, aspecific number (such as three lesions) are selected to be followedbefore the patient is entered in the study.

[0048] A complete response (CR) is the complete disappearance of allmeasurable and evaluable disease, with no new lesions. If the subjecthas effusions, ascites or disease assessable by surgical restaging(e.g., testicular and extragonadal gem cell cancer), the disease must becytologically negative. Patients with markers or indirect evidence ofinvolvement must have normalization of abnormal values. All measurable,evaluable and non-evaluable lesions and sites must be assessed. Apartial response (PR) is found in subjects with at least one measurablelesion with 050% decrease of perpendicular diameters of all measurablelesions, with no progression of evaluable disease, and no new lesions.All measurable and evaluable lesions and sites must be assessed. In lungcancer, a greater than 50% decrease in estimated area of evaluable, butnon-measurable, tumor mass, as agreed upon by two independent observers,not to include pleural effusions. Stabilization is a response that doesnot qualify as a complete response, partial response or progression.

EXAMPLE 3 Treatment of Breast Cancer

[0049] In this example, a 42 year old woman with breast carcinomametastatic to numerous sites in the skeleton received a dose of 11 mcgof calcitriol (Rocaltrol, Roche) administered as 22 tablets (0.5 mcgeach tablet) divided into four nearly equal doses given in hour one,two, three and four. The patient received this same therapy on day 1, 8,15 and 22, and then was observed on study until day 56, and toleratedthe treatment well. She had no Grade II or higher toxicities on the NCItoxicity grading scale (Appendix 2). Subjective beneficial effectsobserved included a reduction in pain and in analgesia required.Objective benefits included a progressive decrease in the serum tumormarker CA15-3 from 445 (pre-treatment) to 365 (day 27), 365 (day 48) and320 (day 70). Radiologic evaluation of areas of known bony involvementshowed progressive sclerosis of multiple lesions in the pelvis and righthip, indicating bone healing as a positive response to therapy. No newlesions or pathologic fractures identified were identified by day 64.

EXAMPLE 4 Treatment of Melanoma

[0050] In this example a 72 year old man with metastatic malignantmelanoma of the right jaw received a dose of 37 mcg of calcitriol(Rocaltrol, Roche) administered as 74 tablets (0.5 mcg each tablet)divided into four nearly equal doses given in hour one, two, three andfour. The patient received this same therapy on day 1, 8, 15 and 22, andobserved until at least day 56. The level of calcitriol in a plasmasample obtained two hours after the last dose of calcitriol (on weekone) was determined using a commercial assay at Endocrine Sciences, Inc.The level was 1826 pg/ml, compared to the range for calcitriol levels innormal controls being 21 to 65 pg/ml. In spite of the markedly elevatedlevels of calcitriol achieved with this weekly schedule, this patientdid not have any subjective or objective toxicity. Levels of serumcalcium and other chemical and hematological parameters in the bloodremained normal.

EXAMPLE 5 Summary of Trial Results

[0051] Patients

[0052] Eligibility criteria included histologically confirmed malignancyrefractory to standard therapy; age ≧18 years; expected survival of >2months; ECOG performance status ≦2; hematocrit ≧30; serum creatinine≦1.2 mg/dL; serum calcium ≦10.5 mg/dL; serum phosphate ≦4.2 mg/dL; ALT≦60 IU/L; total serum bilirubin <2 mg/dL. Exclusion criteria includedpregnancy, history of hypercalcemia, kidney stones, heart failure orsignificant heart disease including myocardial infarction in the last 3months, known cardiac ejection fraction less than 30%, current digoxintherapy, thiazide diuretic therapy within 7 days, bisphosphonatetreatment within 4 weeks, systemic steroid therapy within 2 weeks, andunwillingness or inability to stop all magnesium containing antacids,bile resin binding drugs, or calcium supplements for the duration of thestudy.

[0053] Treatment

[0054] Baseline evaluation included a complete history and physicalexam, complete blood count, serum creatinine, serum calcium, serumphosphate, total serum bilirubin, ALT, alkaline phosphatase, albumin,serum β-hCG in women of childbearing potential, 24 hour urine collectionfor calcium, and tumor measurements.

[0055] Patients were asked to maintain a reduced calcium diet for thefour treatment weeks, with a goal of less than 500 mg per day, asdescribed in Example 1. Calcitriol (Rocaltrol®, Roche Pharmaceuticals)was given orally once a week for four weeks. Each weekly dose was givenin four divided doses given hourly over four hours. The starting dosewas 0.06 μg/kg.

[0056] Monitoring

[0057] Complete blood count, serum creatinine, total serum bilirubin,ALT, alkaline phosphatase were monitored weekly. Serum calcium andphosphate were checked on the treatment day (day 1), and on days 2 and3. Twenty-four hour urinary calcium excretion was measured on day 2. The1,25-dihydroxyvitamin D levels were measured by ¹²⁵I radioimmunoassay(Incstar, Stillwater, Minn.) and by a radioreceptor assay using calfthymus 1,25-dihydroxyvitamin D receptor (Endocrine Sciences, CalabasasHills, Calif.). Peak levels were measured two hours after all the pillshad been ingested. Trough levels were measured approximately 48 hourslater.

[0058] Compliance with the diet was monitored with a seven day dietaryrecall questionnaire directed at calcium rich foods. Daily calciumintake was estimated by adding the calcium content of calcium rich foodsidentified by the questionnaire to the estimated calcium content of thebasal diet. The calcium content of the basal diet was calculated to be 1mg of calcium/9 Kcal. Caloric intake was estimated with the use of theFood Processor 7.0 software (ESHA Research, Salem, Oreg.).

[0059] After completing the four week treatment period, patients weremonitored for four additional weeks. Serum calcium was checked in weeks5 and 7 and tumor measurements were obtained in week 7. All toxicitieswere graded according to NCI Common Toxicity Criteria. Response wasassessed according to WHO guidelines.

[0060] Statistical Considerations

[0061] The planned dose escalation was governed by the multistageescalation scheme described by Gordon and Willson, 1992. The maximaltolerated dose (MTD) was defined as that dose at which ⅓ or fewer of thepatients experienced Grade 3 toxicity (64). Patients who had evidence ofresponse or stable disease, and no Grade 3 or greater toxicity werepermitted to reenroll and receive either the same dose or the nexthigher dose of calcitriol. Statistical analysis was performed usingStatView 5.0 for Windows software (SAS Institute, Cary, N.C.)

RESULTS

[0062] Fifteen different patients were enrolled in 20 cycles of therapy(Table 2). Two patients were withdrawn from the study prior tocompletion of the four week regimen because of disease progression. Nopatient withdrew because of toxicity of therapy or unacceptability ofthe diet. Five patients reenrolled for a second cycle of treatment.TABLE 2 Individual Patients Enrolled on Study Cycle 1 dose Cycle 2 dosePatient Age Gender Malignancy (μg/kg) (μg/kg) 1 79 male Adenocarcinomaof the prostate 0.06 0.12 2 42 female Adenocarcinoma of the breast 0.123 70 male Adenocarcinoma of the lung 0.24 4 72 male Melanoma 0.48 5 53male Squamous Cell of the tonsil 0.48 6 48 female Hepatocellularcarcinoma 0.80 1.60 7 80 male Adenocarcinoma of the prostate 0.96 2.00 853 female Adenocarcinoma of the breast 1.60 9 77 female Adenocarcinomaof the lung 1.92 2.00 10 78 male Adenocarcinoma of the prostate 2.00 1169 male Adenocarcinoma of the prostate 2.00 12 46 female Adenocarcinomaof the breast 2.00 13 47 female Gastrointestinal stromal tumor 2.00 1471 male Adenocarcinoma of the pancreas 2.80 2.80 15 76 maleAdenocarcinoma of the prostate 2.80

[0063] No deaths occurred. No patient withdrew from the study due totoxicity, and no Grade 3 or higher toxicity was seen. All observedtoxicities are listed in Table 3. TABLE 3 Toxicities developed duringeach treatment course (N = 20) Toxicity Grade 1 Grade 2 Grade 3 Grade 4Leukopenia  5¹  1² 0 0 Anemia 3  4² 0 0 Thrombocytopenia 2 0 0 0Hypercalcemia 8 0 0 0 Creatinine elevation 4 0 0 0 Bilirubin elevation 20 0 0 ALT elevation 1 0 0 0 Alkaline phosphatase elevation 2  1² 0 0Nausea and vomiting 5 2 0 0 Diarrhea 3 1 0 0 Constipation 5 0 0 0Dyspepsia 4 0 0 0 Headache 5 0 0 0 Fever 2 0 0 0 Skin rash 1 0 0 0 Boneor muscle pain 8 0 0 0

[0064] The normal range for serum 1,25-dihydroxyvitamin D levels is0.05-0.16 nM (20-65 pg/ml). An approximately linear increase in the peaklevel was observed with increasing dose until the 0.48 μg/kg dose (Table4, FIG. 1). Above this dose, a further elevation of peak levels was notseen. Serum 1,25-dihydroxyvitamin D trough levels returned to normal ornear normal levels by 48 hours (FIG. 1). A limited study of calcitriolpharmacokinetics showed the expected decay in 1,25-dihydroxyvitamin Dlevels after hour 6 (FIG. 2). TABLE 4 Mean Peak and 48 hour1,25-dihydroxyvitamin D levels by dose Dose level (μg/kg) Patients Meanpeak (nM) Mean 48 hour level 0.06 1 0.71 0.27 0.12 2 1.10 0.14 0.24 12.27 0.21 0.48 2 4.11 0.23 0.80 1 3.53 0.96 1 3.83 1.60 2 3.65 1.92 13.34 2.00 6 4.07 0.26 2.80 2 2.96

[0065] Mean serum calcium (normal range 8.5-10.5 mg/dL) increased from9.55 (SD 0.57) mg/dL prior to treatment to 9.76 (SD 0.63) mg/dL 24 hourslater and to 9.88 (SD 0.68) mg/dL at 48 hours (p=0.0002 by a two wayrepeated measures analysis of variance). All calcium levels above thenormal range returned to normal within 2 days with no intervention. Meanserum phosphate (normal range 2.2-4.2 mg/dL) increased from 3.43 (SD0.56) mg/dL prior to treatment to 3.98 (SD 0.57) mg/dL 24 hours laterand dropped to 3.86 (SD 0.53) mg/dL at 48 hours (p<0.0001 by a two wayrepeated measures analysis of variance). Mean 24 hour urinary calciumexcretion (normal range 100-300 mg) increased from 130 (SD 62) mg with arange of 44-292 mg prior to treatment to 263 (SD 125) mg with a range of59-594 on treatment, measured on day 2 of each treatment week (p<0.0001by a one way repeated measures analysis of variance). There was nostatistically significant increase in urinary calcium excretion duringthe treatment period by the Bonferroni/Dunn test.

[0066] Five of eight patients with measurable disease had stabledisease. Among them, an adenocarcinoma of the lung patient, anadenocarcinoma of the pancreas patient, and a hepatocellular carcinomapatient received two cycles of therapy and remained stable for theentire 16 weeks of their time on study. The hepatocellular carcinomapatient had an associated 70% decline in her serum AFP level. Theremaining three patients with measurable disease had evidence ofprogressive disease.

[0067] Four of seven patients without measurable disease had no evidenceof progression. Among them was the breast cancer patient described inExample 3. A prostate cancer patient received two cycles of therapy, andhad a stable PSA for the entire 16 weeks during which the drug wasadministered, in spite of a rapidly rising PSA prior to enrollment. Theremaining three patients without measurable disease had either tumormarker or clinical evidence of progressive disease.

[0068] No patient developed dose-limiting hypercalcemic toxicity fromcalcitriol (<2 mcg/day). Measurements of peak blood calcitriol levels inpatients indicate that blood levels (up to 8.9 nM) are at a level knownto be growth inhibitory for cancer cells in culture. Furthermore, thedrug calcitriol is essentially completely cleared from the blood by day3, and this rapid clearance explains the increased safety profile of theweekly pulse schedule.

[0069] An unanticipated result was the finding that escalation ofcalcitriol dose beyond dose level 0.48 μg/kg did not result in furtherincreases in peak calcitriol levels. More detailed measurement ofcalcitriol levels in one patient (dose level 2.0 μg/kg) indicated thatabsorption is saturated at high doses rather than delayed, as neitherthe peak levels of calcitriol are delayed and the half-life of the drugis not extended beyond the usual time observed in lower dose studies.The maximal tolerated dose (MTD) of calcitriol was not determined by thedata presented in this example.

[0070] In summary, pulsed weekly administration of calcitriol allowssubstantial escalation of the total weekly dose of calcitriol that canbe administered to patients with advanced malignancies. Peak bloodlevels of calcitriol about 25 fold above the upper limit of normal areachieved with minimal toxicity. These levels are well into the rangewhere antiproliferative effects of calcitriol are observed. Based on theobservation that blood levels of calcitriol do not increase linearlywith increased dose beyond the 0.48 μg/kg level, a dose level of 0.5μg/kg is an example of a dose that is therapeutically effective inpatients whose tumor responds to this therapy, but which does not resultin unacceptable hypercalcemia.

EXAMPLE 6 Preparation of Pharmaceutical Dosage Forms

[0071] Preparation of pharmaceutically acceptable compositions of theVitamin D drugs of the present invention can be accomplished usingmethods well known to those with skill in the art. Any of the commoncarriers such as sterile saline solution, plasma, etc., can be utilizedwith the Vitamin D drugs of the invention. Routes of administrationinclude but are not limited to oral, intracranial ventricular (icv),intrathecal (it), intravenous (iv), parenteral, rectal, topicalophthalmic, subconjunctival, nasal, aural and transdermal. The Vitamin Ddrugs of the invention may be administered intravenously in anyconventional medium for intravenous injection such as an aqueous salinemedium. Such medium may also contain conventional pharmaceutical adjunctmaterials such as, for example, pharmaceutically acceptable salts toadjust the osmotic pressure, buffers, preservatives and the like. Amongsuch media are polysorbate, normal saline, lactated Ringer's solution,and plasma. Vitamin D is somewhat insoluble, hence solubilizing agentssuch as sesame oil, or equivalent lipophilic preparations, may be usedto administer the Vitamin D drug.

[0072] Embodiments of the invention comprising medicaments, such astablets or capsules, can be prepared with conventional pharmaceuticallyacceptable carriers, adjuvants and counterions as would be known tothose of skill in the art. The medicaments are preferably in the form ofa unit dose in solid, semi-solid and liquid dosage forms such astablets, pills, powders, liquid solutions or suspensions, and injectableand infusible solutions, for example a unit dose vial. Effective dosageranges included in the unit dose for calcitriol vary from about 5 mcg toabout 100 mcg. The unit dosages of the clacitriol are much higher thanpreviously used, because of the unanticipated finding that high pulse,therapeutically effective doses of the drug can be given withoutinducing symptomatic hypercalcemia.

EXAMPLE 7 Determining Binding Affinity

[0073] Binding affinity of the Vitamin D drugs for the Vitamin Dreceptor can be determined by any acceptable means, such as the VDRbinding analysis and Scatchard plots in Peehl et al., Cancer Research54:805-810, 1994., which is incorporated by reference.

[0074] VDR affinity can be assayed by a competitive receptor assay withradio-labeled calcitriol to determine the Relative Competitive Index(RCI) wherein the RCI for calcitriol is set at 100. The RCI of some ofthe Vitamin D analogs is set forth in Appendix 3.

EXAMPLE 8 Detecting Vitamin D Receptor on Tumor Cells

[0075] The presence of the VDR on tumor cells can be detected by themethods set forth in Peehl et al., which has been incorporated byreference in Example 7. A variety of other assays can be used to detectthe VDR, including immunohistochemistry (Kaiser et al., J. Cancer Res.Clin. Onc 122:356-359, 1996); Western blot (Cross et al., AnticancerResearch 16:2333-2338, 1996); ligand binding assays and DNA probehybridization to RNA (Northern blot) (Endocrinology 132:1952-1960); anddetection of RNA by ribonuclease protection assay (Shabahang et al.,Annals of Surg. Onc. 3:144-149, 1996).

[0076] In view of the many possible embodiments to which the principlesof our invention may be applied, it should be recognized that theillustrated embodiment is only a preferred example of the invention andshould not be taken as a limitation on the scope of the invention.Rather, the scope of the invention is defined by the following claims.We therefore claim as our invention all that comes within the scope andspirit of these claims.

[0077] The numerical values in the table for Relative Competitive Index(RCI) for the nuclear VDR and the RCI for the vitamin D binding protein(DBP) tabulate the data for the indicated parameter in relation to theresult for 1α,25(OH)₂D₃ which is normalized to 100; thus the data valuesrepresent percentages of the 1α,25(OH)₂D₃ value. The key to the dataentries is as follows. The RCI measures the relative ability of ananalog under in vitro conditions to compete with [3H]1α,25(OH)₂D₃ forbinding to the nuclear 1α,25(OH)₂D₃ receptor (VDR) (141) or the plasmavitamin D transport protein (DBP) (142). The cell differentiation dataare related to that for 1α,25(OH)₂D₃ for which the value is set at 1.00.The calcemic index data are set at a value of 1.00 relative to that of1α,25(OH)₂D₃.

[0078] SPC indicates the species of origin of the nVDR where c=chickintestine, r=rat intestine, b=bovine thymus, p=pig intestine, m=MCF-7human breast cancer cells, z=rat osteosarcoma ROS 17/2.8 cells. For theDBP, all data are obtained from the human protein. The celldifferentiation data are obtained in human transformed cell lines whereh=HL-60 cells, u=U-937. The calcemic index is a measure of the relativeability of an analog to generate a “calcemic” response, which is defineddifferently depending upon the assay being conducted; c or i=a measureof intestinal Ca²⁺ absorption (ICA) or bone Ca²⁺ mobilizing activity(BCM) in the vitamin D-deficient chick (146,553); b=bone resorption;cbp=induction of the vitamin D-induced calcium binding protein,calbindin-D_(28k); sc=elevation serum Ca²⁺=concentrations; oc=anincrease in serum osteocalcin levels; r=an increase in urinary Ca²⁺excretion. Reference numbers are indicated in parentheses, and refer toregerence numbers in Boleillon et. al, 1995.

We claim:
 1. A method for the treatment of a hyperproliferative diseasein a subject, wherein the hyperproliferative disease responds totreatment with a Vitamin D drug, comprising administering to the subjecta therapeutically effective pulse dose of the Vitamin D drug in asufficient amount to have an antiproliferative effect, without inducingsevere symptomatic hypercalcemia.
 2. The method of claim 1, comprisingadministering the Vitamin D drug to a subject having a neoplasm thatexpresses a Vitamin D receptor.
 3. The method of claim 2, wherein theneoplasm is selected from the group of cancer of the prostate, breast,colon, lung, head and neck, pancreas, endometrium, bladder, cervix,ovaries, squamous cell carcinoma, renal cell carcinoma, myeloid andlymphocytic leukemia, lymphoma, medullary thryoid carcinoma, melanoma,multiple myeloma, retinoblastoma, and sarcomas of the soft tissues andbone.
 4. The method of claim 3, wherein the neoplasm is breast cancer orprostate cancer.
 5. The method of claim 1, wherein the Vitamin D drughas a calcemic index greater than or equal to calcipitriol.
 6. Themethod of claim 1, wherein the Vitamin D drug has a calcemic indexgreater than 1.0.
 7. The method of claim 5, wherein the Vitamin D drughas a half-life that is no greater than about 1 day.
 8. The method ofclaim 7, wherein the Vitamin D drug has a half-life that is no greaterthan about 6 hours.
 9. The method of claim 8, wherein the Vitamin D drugis administered in an amount that raises a serum level of Vitamin D inthe subject with a tumor to a supraphysiologic amount for a sufficientperiod of time to induce differentiation or regression of the tumorwithout causing symptomatic hypercalcemia.
 10. The method of claim 1,wherein the Vitamin D drug is calcitriol, which is administered in atherapeutically effective pulse dose no more than once every three days.11. The method of claim 10, wherein the calcitriol is administeredorally in a dose of at least 0.12 mcg/kg per day no more than once perweek.
 12. The method of claim 10, wherein the calcitriol is administeredorally in a dose of at least 0.48 mcg/kg or about 1 mcg/kg per day nomore than once per week.
 13. A method of treating a tumor in a subject,wherein the tumor expresses a Vitamin D receptor and is responsive totreatment with a Vitamin D drug, the method comprising administeringorally to the subject, no more than once every three days, a dose ofcalcitriol of about 0.5 mcg/kg.
 14. The method of claim 13, wherein theVitamin D drug is administered to the subject no more than once perweek.
 15. A composition comprising a Vitamin D drug in a pharmaceuticaldosage form in a dosage that would cause hypercalcemia is administereddaily.
 16. The composition of claim 15, wherein the Vitamin D drug iscalcitriol, contained in the composition in an amount of at least 5 mcg.17. The composition of claim 16, wherein the pharmaceutical dosage formis an oral dosage form containing at least 100 mcg per unit of the oraldosage form.
 18. The composition of claim 16, wherein the pharmaceuticaldosage form is a tablet or capsule.
 19. The composition of claim 16,wherein the Vitamin D drug is calcitriol, and the pharmaceutical dosageform is a tablet containing at least 5 mcg calcitriol.
 20. The method ofclaim 1, wherein the subject is prescribed a reduced calcium diet for asufficient period of time prior to administration of the Vitamin D drugto reduce absorption of dietary calcium.
 21. A method of treating in asubject a tumor that expresses a Vitamin D receptor, the methodcomprising raising a blood level of Vitamin D to a sufficientlysupraphysiologic level for a sufficient period of time to inhibit growthof the tumor, without inducing hypercalcemia in the subject.
 22. Themethod of claim 21, wherein the blood level of Vitamin D is raised byadministering a Vitamin D drug to the subject.
 23. The method of claim22, wherein the Vitamin D drug is calcitriol.
 24. The method of claim23, wherein the calcitriol is administered in a dose of about 0.50mcg/kg once per week.